analytical bioc lec 1

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Questions and Answers

What factor significantly affects protein solubility?

Protein structure

Amino acid sequence

Buffer conditions (correct)

Temperature

What may happen if protein complexes are diluted to concentrations around their effective dissociation concentration?

They will remain stable and functional.

They will merge with other complexes.

They may dissociate and lose biological activity. (correct)

They will aggregate and form larger complexes.

How can protein adsorption be minimized when using biochemical equipment?

By lowering the temperature during the experiment.

By using higher concentrations of protein.

By utilizing materials that proteins find less sticky. (correct)

By increasing the pH of the protein solution.

What is a common consequence of air being non-polar when in contact with proteins?

Proteins may denature at the air-liquid interface. Signup and view all the answers

Which of the following methods can help reduce protein adherence to surfaces?

Adding detergents or carrier proteins. Signup and view all the answers

What type of mechanical force is likely to cause protein denaturation?

Shear forces through a needle Signup and view all the answers

At what concentration are most long-term protein complexes stabilized?

1 nM Signup and view all the answers

What happens to proteins under extreme pressure (>5 k atm)?

They remain unaffected. Signup and view all the answers

What is the typical salt concentration maintained in cellular environments?

150 mM Signup and view all the answers

Which type of compounds are known for stabilizing proteins but also have the ability to precipitate them?

Kosmotropes Signup and view all the answers

Which of the following salts tends to denature proteins and also dissolve them?

Thiocyanate Signup and view all the answers

What is the primary effect of chaotropes on proteins?

Decrease the hydrophobic effect Signup and view all the answers

What effect do kosmotropes have on the hydrophobic effect?

They increase it Signup and view all the answers

Which of the following is an example of a common chaotropic agent?

Urea Signup and view all the answers

What discovery did Hofmeister make about salts and their effects on proteins?

Salts have varying impacts based on their solvation energies. Signup and view all the answers

What happens to proteins as temperatures increase?

They unfold irreversibly. Signup and view all the answers

Why is it advisable to work with proteins cold but not frozen?

Freezing can cause irreversible damage to proteins. Signup and view all the answers

How do sulfate salts affect proteins according to Hofmeister's findings?

They stabilize proteins and also precipitate them. Signup and view all the answers

What is a consequence of freezing proteins?

Ice crystal growth that damages the protein structure. Signup and view all the answers

Which method is used for rapid freezing of protein solutions to minimize ice crystal growth?

Ethanol + dry ice or liquid nitrogen baths. Signup and view all the answers

What are cryoprotectants used for in the freezing process?

To reduce damage during freezing. Signup and view all the answers

Which of the following is a common cryoprotectant used for proteins?

Glycerol. Signup and view all the answers

What is a recommended best practice for freezing protein solutions?

Freeze small, single-use samples. Signup and view all the answers

Human diacylglycerol kinase is noted for being:

Very sensitive to freezing. Signup and view all the answers

Which amino acid is observed to be the least abundant in vertebrates?

Tryptophan (W) Signup and view all the answers

How does the presence of water affect the structure of proteins?

It stabilizes exposed polar side chains. Signup and view all the answers

What is the average molecular weight contribution of each amino acid in proteins?

110 Da Signup and view all the answers

Which type of amino acids are more commonly found in the exposed surface of proteins?

Polar amino acids Signup and view all the answers

What happens to proteins when they become partly unfolded?

They may refold if they are smaller. Signup and view all the answers

Which characteristic of proteins contributes to their marginal stability?

Significant number of weak interactions. Signup and view all the answers

What can significantly disrupt the normal structure of dissolved proteins?

Inorganic solvents and high salt concentrations. Signup and view all the answers

What role do charged residues play in amino acid abundance?

They are slightly enriched in protein compositions. Signup and view all the answers

What is a key requirement for choosing a biological buffer?

It should minimize the effect outside its pKa range. Signup and view all the answers

Which of the following is a characteristic of 'Good's buffers'?

They are temperature sensitive. Signup and view all the answers

Which buffer would be most appropriate for use at pH 7.5?

HEPES (pKa 7.5) Signup and view all the answers

What effect does lowering the pH have on proteins with neutral His residues?

They become protonated. Signup and view all the answers

At what pH range is acid denaturation of proteins generally observed?

pH 2-5 Signup and view all the answers

What role do salts play in the stabilization of proteins in solution?

They shield charged side chains. Signup and view all the answers

What defines the pKa of a buffer?

The pH at which the concentration of acid and conjugate base are equal. Signup and view all the answers

Which of the following buffers has a pKa closest to 8.0?

TRIS (pKa ~8.1) Signup and view all the answers

Define accuracy

how close measured value is to the true value Signup and view all the answers

Define precision

how similar the readings produced by the repeated experiments are Signup and view all the answers

Which method is a binding-based separation technique?

Chromatography Signup and view all the answers

Which technique is most appropriate for separating charged particles based on their size?

Electrophoresis Signup and view all the answers

Which type of spectroscopy is primarily used for analyzing the absorption of UV and visible light?

UV-visible Spectroscopy Signup and view all the answers

What technique can identify the mass-to-charge ratio of ions?

Mass Spectrometry Signup and view all the answers

Which of the following methods would be considered a physics-based separation technique?

Electrophoresis Signup and view all the answers

what role do IUPs play

play a role in cell signaling and protein interactions Signup and view all the answers

what type of error is this? This happens when a test incorrectly indicates the presence of a condition when it is not actually there.

false positive Signup and view all the answers

what type of error is this? This occurs when a test fails to detect a condition that is actually present.

false negative Signup and view all the answers

Which amino acids are categorized as polar?

Arg, Lys, His, Asp Signup and view all the answers

What are the properties of residues that make them significant in analytical biochemistry?

They absorb light in the UV-visible range. Signup and view all the answers

Which amino acid is not included in the list of especially important amino acids for analytical biochemistry?

Ser Signup and view all the answers

Which group of amino acids is considered non-polar?

Trp, Phe, Met, Val Signup and view all the answers

Which statement accurately describes the importance of certain residues in proteins?

They carry charges and can act as metal binders. Signup and view all the answers

What is the main charge state of guanidine groups in arginine under physiological pH conditions?

Positively charged Signup and view all the answers

Which amino acid has a pKa value that allows it to remain positively charged at slightly acidic pH levels?

Histidine Signup and view all the answers

What property does the thiol side chain of cysteine confer that is crucial for biochemical labeling?

Highly reactive nucleophile Signup and view all the answers

Which of the following statements is true regarding the absorption properties of tryptophan and tyrosine?

Tryptophan fluoresces, whereas tyrosine does not Signup and view all the answers

What is the consequence of deprotonation of cysteine's thiol group?

Acquisition of a negative charge Signup and view all the answers

At what pH does the side chain of tyrosine typically lose its proton, creating a phenolic anion?

pH 10 Signup and view all the answers

Which amino acid side chain can bind metal ions, enhancing its utility in affinity purification methods?

Histidine Signup and view all the answers

Which statement is true about the charge states of aspartate and glutamate under neutral pH conditions?

Both are negatively charged Signup and view all the answers

What is the typical range of purified protein concentrations used for biochemical experiments?

0.1 – 50 mg/mL Signup and view all the answers

What is a common concentration of total protein found in cells?

400 mg/mL Signup and view all the answers

For enzyme assays, what is an example of a typical protein concentration that may be used?

~1 μg/mL Signup and view all the answers

Which concentration condition is likely to cause proteins to precipitate?

Below 10 μg/mL for certain proteins Signup and view all the answers

How can poorly soluble proteins be made more soluble?

By optimizing the buffer conditions Signup and view all the answers

Which statement is true regarding the solubility of individual proteins in pure form?

Individual proteins tend to be less soluble when pure. Signup and view all the answers

What is the primary process that occurs during lyophilization?

Sublimation of water from the solid phase, leaving solutes behind Signup and view all the answers

Which statement accurately describes the stability of proteins after lyophilization?

Many proteins can remain stable for years if kept cold after lyophilization. Signup and view all the answers

What can facilitate the reactivation of a lyophilized protein?

Rehydrating the protein with water Signup and view all the answers

In which industries is lyophilization most likely utilized?

Commercial sale of enzymes and pharmaceuticals Signup and view all the answers

What is a critical aspect of lyophilization that may need optimization for proteins?

The conditions under which the proteins are dried Signup and view all the answers

Study Notes

Amino Acids and Their Abundance

Amino acids have different observed frequencies in vertebrates.
Some amino acids are relatively rare (e.g. Cys, Tyr, His, Trp), while others are more common.
Charged residues are slightly enriched.
The average weight of an amino acid in a protein is 110 Da.

Protein Structure Organization

Non-polar amino acids are buried in the core of a protein.
Polar residues on the surface interact with water.
This organization is generally essential for protein function.

Water's Role in Protein Stability

Water molecules are bound by hydrogen bonds on a protein's surface.
Some water molecules are buried inside the protein structure.
Water stabilizes polar side chains, charged groups and ions.
Proteins require hydration to maintain their structure.
Dehydration, freezing, inorganic solvents, and high salt concentrations can disrupt water structure, potentially destabilizing proteins.

Protein Structure Stability

Protein stability is achieved by a significant number of weak interactions (e.g. van der Waals forces and hydrogen bonds) alongside the hydrophobic effect.
Proteins are only marginally stable, exhibiting dynamics.
This enables functional flexibility but also makes them susceptible to unfolding.
Heat can cause protein unfolding.

Protein Unfolding and Aggregation

Unfolded proteins can refold, with smaller proteins exhibiting greater refolding capabilities.
Unfolding exposes hydrophobic core residues, leading to aggregation and potentially precipitation.
Protein solubility is influenced by factors including pH, salt concentration, and other solutes.
Optimizing buffer conditions can improve the solubility of poorly-soluble proteins.

Protein Complexes and Dilution

Dilution of protein complexes to concentrations below their effective dissociation concentration can lead to dissociation.
Co-factors may also dissociate from proteins with excessive dilution.
Protein complexes can be stabilized by sub-nanomolar interactions, with most maintaining function at 1 nM concentration.

Protein Adsorption

Proteins have both polar and non-polar surfaces, often "pre-organized" for binding.
This allows proteins to adhere to various surfaces.
Biochemical equipment and disposables are typically made from materials less "sticky" to proteins (e.g. glass, steel, non-polar plastics, polysaccharides).
Detergents, increased salt concentrations, and carrier proteins (e.g. BSA) can minimize adherence.
Adsorption is more pronounced with dilute protein solutions.

Air and Protein Structure

Air is very non-polar and cannot form hydrogen bonds.
Proteins can unfold at the air-liquid interface.
An example is protein denaturation in beaten egg whites, creating a stable foam.
Avoid excess air-water surface area and bubble formation in biochemical experiments.

Mechanical Forces and Protein Structure

Proteins can be denatured by very high pressures, but are generally resistant to pressures encountered in biochemical labs.
Shear forces can unfold proteins.
For example, proteins can denature when passing through narrow needles.

Proteins and Temperature Stability

Higher temperatures increase solvent kinetic energy and can disrupt protein stability.
Increasing temperatures can irreversibly unfold proteins.
Most proteins tolerate temperatures up to 37°C, but tolerance varies.
Proteins evolved in high temperatures may function sub-optimally at lower temperatures.
It is generally recommended to work with proteins cold (on ice) but not frozen.
Protein assays are typically conducted between 4°C (ice) and 37°C (body temperature).

Freezing and Thawing Proteins

Freezing is generally damaging to proteins.
Ice crystals of pure water grow during freezing, concentrating solutes and proteins in remaining liquid. This leads to high salt/solute/protein concentrations and altered pH, potentially causing protein aggregation and inactivation.
Freezing also exerts mechanical stress due to water expansion.
Repeated freeze-thaw cycles should be avoided.

Flash Freezing: A Storage Strategy

Rapid freezing prevents ice crystal growth, forming glass-like vitreous ice that incorporates impurities like proteins.
Ethanol/dry ice or liquid nitrogen baths facilitate rapid freezing.
Small, single-use samples are best for minimizing protein damage.
Proteins needed for the day can be thawed on ice with excess discarded at the end of the day.

Cryoprotectants

Cryoprotectants reduce (but rarely eliminate) damage during freezing.
They are solutes, like sugars and poly-alcohols, that mimic water in forming hydrogen bonds and prevent ice crystal growth.
Glycerol, ethylene glycol, and trehalose are commonly used.
High concentrations can keep solutions liquid below 0°C.

Sensitivity to Freezing: Diacylglycerol Kinase

Diacylglycerol kinase is very sensitive to freezing.

Biological Buffers

Buffers are chosen with a pKa close to the target pH.
Buffers exert minimal effect beyond 1 pH unit away from their pKa.
Biochemical buffers should be non-toxic, highly soluble, not absorb UV-visible light, not bind metals, and be chemically unreactive.
Simple chemical structures are preferred.
Most buffers are temperature sensitive.
Adjust the pH of buffers at their intended use temperature.

Common Biochemical Buffers

MES (pKa 6.2), TRIS (pKa ~8.1), HEPES (pKa 7.5), Bis TRIS (pKa ~6.5), TAPS (pKa ~8.4) are known as "Good's buffers".
These buffers use amine groups as the conjugate base.
Charge stabilizing groups (e.g. hydroxyl or sulfonates) affect the pKa.
Varying chemical structures yields buffers with diverse pKas.
Acetate, citrate, phosphate, and bicarbonate ions are also used in specific contexts.

Acid/Base Protein Denaturation

As pH decreases, neutral His residues become protonated. Asp and Glu become protonated, neutralizing their charges. The protein becomes positively charged, leading to destabilizing charge-charge repulsion.
At high pH, His, Lys, and eventually Arg become neutralized, while Cys, Tyr, Asp, and Glu become negatively charged. This also leads to charge-charge repulsion and protein destabilization.
Extreme pH denatures proteins.
Acid denaturation occurs around pH 2-5, base denaturation above pH 10.
The specific pH for protein inactivation/unfolding varies.
Some enzymes (e.g. stomach enzymes) are acid-stable.

Salts and Protein Stability

Salts (paired anions and cations) contribute ionic strength to solutions.
These ions associate with charged protein side chains, promoting solubility and shielding charges.
Cellular salt concentrations are around 150 mM (mostly NaCl).
Salt is usually added to stabilize proteins in solutions.
Low salt concentrations can promote protein precipitation.
High salt concentrations can also precipitate proteins, depending on the salt.

Hofmeister Series

Hofmeister (1888) discovered that different salts have diverse effects on proteins.
Sulfate salts tend to stabilize proteins and promote precipitation.
Thiocyanate tends to denature proteins and facilitate dissolution.
These differences are attributed to changes in solvation energies of ions, which in turn affect the hydrophobic effect.

Chaotropes & Kosmotropes

Chaotropes decrease the hydrophobic effect, promoting protein dissociation and denaturation/unfolding.
Examples include organic molecules like ethanol, butanol, urea and guanidinium.
Chaotropes are used to unfold proteins, e.g. to stop enzymatic reactions.
Kosmotropes increase the hydrophobic effect, encouraging protein folding and interactions.
They promote protein stability and facilitate interactions.

Protein Solubility

Protein solubility is significantly affected by hydrophobic interactions, ionic strength, and pH.
Diluting protein complexes to their effective dissociation concentration can disrupt their structure and lead to dissociation.

Minimizing Protein Adsorption

Hydrophobic interactions are a major cause of protein adsorption to surfaces.
Minimizing air contact with protein solutions is crucial, as air is non-polar and can lead to surface tension and adsorption.
Using wettable surfaces and reducing surface area are helpful strategies to minimize adsorption in biochemical equipment.

Protein Denaturation

Mechanical force can disrupt protein structure and lead to denaturation.
Extreme pressure (>5 k atm) can cause protein denaturation due to structural changes.

Protein Stabilization

Salt concentration in cellular environments is typically low, preventing protein precipitation.
High salt concentrations can both stabilize and precipitate proteins, depending on the specific salt and protein involved.
Chaotropes disrupt protein structure by weakening hydrophobic interactions.
Kosmotropes strengthen the hydrophobic effect, promoting protein folding.
Sulfate salts have a stabilizing effect on proteins according to Hofmeister's findings.

Protein Freezing

Freezing proteins can lead to structural damage due to ice crystal formation.
Rapid freezing methods, like flash freezing with liquid nitrogen, minimize ice crystal growth.
Cryoprotectants are used to protect proteins during freezing by lowering the freezing point and reducing ice crystal formation.

Protein Structure and Amino Acid Composition

Water plays a crucial role in maintaining the structure of proteins.
Charged residues are more commonly found on the surface of proteins, contributing to their solubility.
Marginal stability is a key characteristic of proteins, making them susceptible to environmental changes.
Unfolding can disrupt the normal structure of proteins, making them less functional.

Buffers and pH

Biological buffers are essential for maintaining a stable pH in protein solutions.
Good's buffers are preferred due to their non-toxic nature and good buffering capacity over a wide pH range.
Lowering the pH can lead to protonation of neutral His residues, affecting protein structure and function.

Separation Techniques

Binding-based separation techniques rely on specific interactions between the target molecule and a stationary phase.
Size exclusion chromatography is appropriate for separating charged particles based on size.
UV-Vis spectroscopy is used to analyze the absorption of UV and visible light by molecules.
Mass spectrometry identifies the mass-to-charge ratio of ions.
Physics-based separation techniques utilize physical properties like size, charge, or density to separate molecules.

Amino Acid Classification

Amino acids can be categorized into polar and non-polar groups based on their side chains' interactions with water.
Polar amino acids have side chains that interact with water due to their charged or polar nature.
Non-polar amino acids have side chains that are hydrophobic and do not interact well with water.

Important Amino Acids in Analytical Biochemistry

Eight amino acids are crucial in analytical biochemistry due to their unique properties: arginine (Arg), lysine (Lys), glutamic acid (Glu), aspartic acid (Asp), tryptophan (Trp), tyrosine (Tyr), cysteine (Cys), and histidine (His).
These key amino acids possess characteristics like carrying charges, engaging in chemical reactions, having strong affinity for metals, and potentially absorbing or fluorescing light in the UV-visible spectrum.

Aspartic Acid and Glutamic Acid

Aspartic acid (Asp, D) and Glutamic acid (Glu, E) both have carboxyl groups with a negative charge
Asp has one methylene group, Glu has two
The carboxyl group can be neutralized under strongly acidic conditions
The pKa is approximately 4.5

Positively Charged Amino Acids

Arginine (Arg, R) and Lysine (Lys, K) are positively charged
Arginine has a guanidine group which is only deprotonated at a very basic pH, about 12.5, meaning it is almost always positively charged
Arginine’s guanidine group is positively charged, but is deprotonated at strongly basic pH
Lysine’s amine group is positively charged, except at strongly basic pH
Lysine's amine group is also present on the N-terminus of a polypeptide
Lysine's amine group can react specifically with isothiocyanate

Histidine

Histidine (His, H) is positively charged at slightly acidic pH
Histidine contains an imidazole ring
The protonated imidazole gives it a +1 charge, while the deprotonated form is neutral
Histidine’s pKa is approximately 6.5
Histidine has a good affinity for metal ions, which is useful for metal ion affinity purification

Aromatic Amino Acids

Tyrosine (Tyr, Y) and Tryptophan (Trp, W) are aromatic amino acids
Tyrosine has an OH group which can be deprotonated, creating a phenolic anion
Tyrosine absorbs UV light, but less effectively than tryptophan
Tyrosine does not fluoresce
Tryptophan cannot be ionized
Tryptophan absorbs UV light strongly and re-emits it strongly as fluorescence
Tryptophan is the rarest amino acid
Tyrosine’s pKa is approximately 10

Cysteine

Cysteine (Cys, C) has a thiol side chain
Cysteine can be deprotonated, giving it a negative charge
Cysteine’s thiol group is the most nucleophilic part of a protein
This allows for chemical labeling experiments
Cysteine is usually unreactive when buried in the core of a protein
Two cysteine molecules can react to form a disulfide bond (cystine)
Cystine absorbs UV light weakly
Cysteine is relatively rare in proteins

Side Chains and pKa values

Side chain pKa values can be influenced by the local environment within a protein
Positively charged residues will stabilize a nearby negatively charged side chain
Non-polar environments favor the neutral state of a side chain
These factors can alter the pKa of a residue
For example, Glu172 in xylanase has a measured pKa of 6.8, which is higher than the standard 4.5 pKa value
This is due to the destabilization of a negative charge on Glu172 by adjacent residues
Standard pKa values represent averages, and specific residues in a protein can have pKa values shifted by 1-2 pH units.

Protein Concentrations for Experiments

Purified proteins are typically prepared at concentrations ranging from 0.1 to 50 milligrams per milliliter (mg/mL).
Individual experiments using purified proteins, like enzyme assays, may employ very small amounts, often around 1 microgram per milliliter (μg/mL).
The total protein concentration within cells is considerably higher, averaging approximately 400 mg/mL.